The long-term objective of this research program is to develop novel types of phosphoramidate-based anticancer drugs and investigate their mechanisms of action. This drug development program is based upon the design and synthesis of prodrug molecules containing a non-cytotoxic phosphoramidate ester that undergo enzyme-catalyzed activation via cleavage of the ester bond to liberate the highly cytotoxic phosphoramidate anion. A series of bioreductive alkylating agents was developed previously that are highly toxic to tumor cell lines in vitro and have potent antitumor activity in vivo. This strategy will now be extended to the design and synthesis of compounds that can be activated by DT-diaphorase, an enzyme that is overexpressed in a number of human solid tumors, or by beta-lactamase, an enzyme that is used in antibody-directed enzyme prodrug therapy.
Specific aims are to: 1) Develop a 3-dimensional structure-function relationship for human DT-diaphorase to provide a model for the rational design of new drugs; 2) Design, synthesize and evaluate novel phosphoramidate analogs that are activated selectively by the enzyme DT-diaphorase; 3) Design, synthesize and evaluate novel cephalosporin phosphoramidates that are activated selectively by beta-lactamase; 4) Evaluate all new agents for activation and cytotoxicity in vitro, and evaluate selected compounds for antitumor activity in vivo. Purified enzyme preparations will be used to measure the activation of these compounds, and clonogenic assays in B16, MCF-7, HT-29 and BE cell lines will be used to assess cytotoxicity. B16, HT-29 and MX-1 models will be used to evaluate antitumor activity in vivo. The ultimate goal is to exploit the unique properties of phosphoramidate chemistry to develop new drugs with reduced host toxicity and increased efficacy against resistant poorly responsive tumors.
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